1. Field:
This invention is concerned generally with identifying protein-ligand interactions, and specifically with peptide ligands which bind alpha-1 proteinase inhibitor (AlPI) and which may be used in a method for the affinity purification of AlPI.
2. Prior Art:
AlPI is a (gycoprotein with a molecular weight of 53,000 Daltons. AlPI is an inhibitor of proteases such as trypsin, chymotrypsin, and elastase. A current therapeutic use of AlPI is the inhibition of lymphocyte elastase in the lungs. When AlPI is not present in sufficient quantities to regulate elastase activity due to genetic deficiency, the elastase breaks down lung tissue, resulting in lung tissue damage and emphysema. AlPI replacement therapy has been successfully used for treatment of this form of emphysema.
While the AlPI gene has been transformed and expressed in microorganisms, cell lines, and sheep, a satisfactory recombinant product has yet to be produced. Human plasma is currently the only approved source of therapeutic AlPI. To date a practical process which gives both high yield and high purity AlPI has not been available.
Various methods of purifying AlPI from human plasma have been described. Bollen et al., U.S. Pat. No. 4,629,567 (1986) used five different chromatography steps to purify AlPI from yeast, E. coli, and human plasma. The five steps involved DEAE ion exchange, thio-disulfide exchange, heparin affinity, zinc-chelate chromatography, and amino hexyl ion exchange. No purity and yield data were shown.
Novika et al. (1989) reported isolation methods from the by-products of the manufacture of blood products. They used affinity, DEAE cellulose, and gel filtration chromatographies. The purity and yield data were not available.
Podiarenc et al. (1989) reported a single step procedure for isolation of AlPI from human plasma using affinity chromatography with monoclonal antibodies. AlPI activity was increased 61.1 fold with a yield of 20%.
Burnouf et al. (1987) starting with plasma supernatant A (equivalent to Cohn Fraction II+III) used DEAE chromatography and size exclusion chromatography to produce an AlPI which was 80-90% pure (by SDS-PAGE) with a 36-fold increase in purity. Recovery was 65-70% from the supernatant A.
Hein et al. (1990) presented a process which employs Cohn Fraction IV-1 as the starting, material and utilized fractional precipitation with polyethylene glycol followed by anion exchange chromatography on DEAE Sepharose.RTM.. The final product has a purity of about 60% with 45% yield. Other similarly based purifications from Cohn Fractions I, II+III and IV-1 are presented in U.S. Pat. Nos. 4,379,087 (1983), 4,439,358 (1984), and 4,697,003 (1987) by Coan et al. and U.S. Pat. No. 4,656,254 (1987) by Shearer et al.
Dubin et al. (1990) have shown a two step chromatographic purification. First AlPI, C1 inhibitor, alpha-1 antichymotrypsin, and inter alpha-1 trypsin inhibitor were eluted from Blue Sepharose.RTM. and the AlPI was purified by gel filtration. Purity and yield data were not available.
Ballieux et al. (1993) purified an AlPI and proteinase-3 complex from purulent sputum using 4-phenlybutylamine affinity chromatography, cation exchange, and a final immunoaffinity step. The pH of the buffer used in the cation exchange step was 7.0. Under the conditions used, most of the sputum proteins bound to the resin, but AlPI and proteinase-3 passed through without binding.
Lebing et al., U.S. Pat. No. 5,610,285 (1997) purified AlPI from Cohn Fractions IV-1 and II+III by changing the solution conditions to low ionic strength and pH 6.0. Passing the mixture over a cation exchange chromatography resin bound other proteins, but allowed a purified fraction of AlPI to flow through the column.
Hwang et al., U.S. Pat. No. 5,616,693 (1997) purified AlPI from Cohn Fraction IV-1. The fraction was precipitated with polyethylene glycol to remove other proteins. Zinc chloride was then added to the supernatant to precipitate a crude AlPI fraction. The crude AlPI fraction was resolubilized and purified using an anion exchange chromatography step.